Nonlytic exocytosis of Cryptococcus neoformans from neutrophils in the brain vasculature
Cryptococcus neoformans (C. neoformans) is an encapsulated budding yeast that causes life-threatening meningoencephalitis in immunocompromised individuals, especially those with acquired immunodeficiency syndrome (AIDS). To cause meningoencephalitis, C. neoformans circulating in the bloodstream must first be arrested in the brain microvasculature. Neutrophils, the most abundant phagocytes in the bloodstream and the first leukocytes to be recruited to an infection site, can ingest C. neoformans. Little is known about how neutrophils interact with arrested fungal cells in the brain microvasculature.
A blood-brain barrier (BBB) in vitro model was established. The interactions between neutrophils adhering to brain endothelial cells and fungi were observed under a live cell imaging microscope. A flow cytometry assay was developed to explore the mechanisms. Immunofluorescence staining of brain tissues was utilized to validate the in vitro phenomena.
Using real-time imaging, we observed that neutrophils adhered to a monolayer of mouse brain endothelial cells could expel ingested C. neoformans without lysis of the neutrophils or fungi in vitro, demonstrating nonlytic exocytosis of fungal cells from neutrophils. Furthermore, nonlytic exocytosis of C. neoformans from neutrophils was influenced by either the fungus (capsule and viability) or the neutrophil (phagosomal pH and actin polymerization). Moreover, nonlytic exocytosis of C. neoformans from neutrophils was recorded in brain tissue.
These results highlight a novel function by which neutrophils extrude C. neoformans in the brain vasculature.
KeywordsC. neoformans Neutrophils Brain Nonlytic exocytosis real-time imaging
Acquired immunodeficiency syndrome
- C. neoformans
Human immunodeficiency virus
Cryptococcosis is an acquired immunodeficiency virus (AIDS)-defining opportunistic infection that also occurs in organ transplant recipients and cancer patients . Globally, it is estimated that there are an estimated 223,100 cases of cryptococcosis and approximately 181,100 deaths from human immunodeficiency virus (HIV)-associated cryptococcal disease . Additionally, cryptococcosis is also an infrequent fungal infection in patients with systemic lupus erythematosus  or tuberculosis [4, 5]. The causative microorganism is the encapsulated fungus Cryptococcus neoformans. Fungal cells are believed to enter the body through the respiratory tract and initially cause pneumonia. The most devastating event occurs when fungi disseminate into the brain via the bloodstream, consequently resulting in fatal meningoencephalitis.
Fungemia is often detected in AIDS patients during cryptococcosis . Although extrapulmonary dissemination appears to be macrophage-associated , free fungal cells have been detected in the bloodstream . These circulating fungal cells could be directly derived from the lung as free fungus or released from macrophages, as recent evidence has shown that macrophages can expel ingested C. neoformans via nonlytic exocytosis [9, 10, 11].
Recently, brain invasion by C. neoformans has been visualized in a mouse model based on intravital microscopy, and an important series of events that occur prior to transmigration into the brain has been postulated [12, 13]. The critical steps include fungal arrest in the vasculature of the brain and interaction and signaling of the fungal and endothelial cells leading to transmigration [12, 13]. As a result of these processes, the arrested fungal cells remain within the brain vasculature for hours, providing the opportunity for immune cells circulating in the bloodstream to recognize the fungal cells. However, the intravascular interactions of immune cells with the arrested fungal cells are largely unknown. This knowledge gap limits our advances in the prevention and treatment of the illness.
Neutrophils, one of the major players during infection, are typically the first immune cells to be recruited to an infection site and are capable of eliminating microorganisms by multiple means . There is evidence that neutrophils play roles in protecting the host against C. neoformans . In vitro, neutrophils internalize C. neoformans following opsonization with complement and antibody [16, 17, 18]. In vivo, neutrophils ingest C. neoformans in the lungs of mice following intratracheal infection  and kill the fungi with the aid of complement C5a in the brain vasculature . Previously, we reported that neutrophils may ingest and remove C. neoformans from brain vessels . In this study, we recorded neutrophils expelling C. neoformans without causing lysis of either the neutrophils or fungi, which may be regulated by fungal virulence and neutrophil actin polymerization.
Materials and methods
C57BL/6 J mice from the Laboratory Animal Center, Yangzhou University (Yangzhou, China) were housed under a 12 h light/dark cycle in specific pathogen-free conditions with free access to mouse chow and water. Animal experiments were approved by the Laboratory Animal Ethics Committee of Nanjing Medical University (1708004).
C. neoformans strain H99 was obtained from ATCC (catalog number 208821). C. neoformans B3501, Cap67, and Cap59 strains were gifts from Dr. Min Chen from the Second Military Medical University. The organisms were cultured in Sabouraud’s dextrose broth (Difco) at 32 °C with gentle rotation for 16 h and then washed 3 times in sterile PBS (pH 7.4) before use.
Nonlytic exocytosis in vitro observed under a confocal microscope
Neutrophils were purified from bone marrow cells using a Percoll density gradient . To determine the purity of neutrophils isolated from the bone marrow, the isolated cells were stained with PE anti-mouse F4/80 mAb (clone BM8, eBioscience) and Alexa Fluor 647 anti-mouse Gr-1 mAb (clone RB6-8C5, Invitrogen). As a control, cells were incubated with the corresponding isotype control antibodies. Antibodies and their ligands are listed in Additional file 1: Table S1.
To observe the dynamic interactions of neutrophils with the ingested C. neoformans, neutrophils purified from bone marrow cells were incubated with Alexa Fluor 647 anti-Gr-1 mAb (RB6-8C5, Invitrogen) and PE anti-mouse F4/80 mAb (BM8, eBioscience) for 30 min. The prestained neutrophils (5 × 106) and C. neoformans labeled with FITC (5 × 107) were added to the surface of a monolayer of mouse brain endothelial cells bEnd.3 (CRL-2299, ATCC) in a glass-bottom dish (P35G-1.5-10, MatTek), which represented an in vitro BBB model . E1 mAb specific for cryptococcal polysaccharide (a gift from Dr. F. Dromer, Paris, France) was added to the culture, and the cells were incubated for 1 h in the live cell culture equipment of the confocal microscope (LSM510, Zeiss). The dish was then extensively washed 3 times with complete medium to remove most of the nonadherent neutrophils and C. neoformans. The interactions of neutrophils with the ingested C. neoformans were imaged, and time-lapse videos were taken using a confocal microscope.
Flow cytometry analysis of nonlytic exocytosis
Flow cytometry analysis of nonlytic exocytosis was first developed by André Moraes Nicola and colleagues . Briefly, neutrophils were purified from bone marrow  with a neutrophil isolation kit (130–097-658, Millipore) and stained with DDAO-SE (0.5 μM, Invitrogen) in PBS for 10 min and washed 3 times. C. neoformans were stained with FITC (1 mg/ml, Sigma-Aldrich) in PBS for 15 min and washed 3 times. Furthermore, neutrophils were stained with 7-AAD (1 μg/ml, Invitrogen) for 5 min to detect cell death, and C. neoformans were stained with Uvitex 2B (0.01%, Polysciences Inc.) in PBS for 1 min to detect fungi outside of neutrophils (Additional file 2: Figure S1). DDAO-SE-stained neutrophils and FITC-stained H99 (neutrophils:H99 = 1:1) cells were incubated in the presence of the C. neoformans-specific anti-E1 antibody (1 μg/ml) for 2 h and further stained with Uvitex-2B and 7-AAD. Subsequently, FITC+DDAO-SE+ 7-AAD−Unvitex2B− cells, which represented live neutrophils containing C. neoformans, were flow sorted with a BD FACS ARIA II SORP. Immediately after sorting, the cells were analyzed for purity, and 1 × 106 sorted neutrophils in 1 ml of complete medium (10% fetal calf serum in RPMI 1640 medium) were incubated in the well of a 24-well plate with or without chemicals for 2 h. After incubation, the cells were harvested and stained with 7-AAD again for flow cytometry analysis. FITC−DDAO-SE+ 7-AAD− cells were considered live neutrophils that had expelled the ingested C. neoformans. As described by Moraes Nicola and colleagues , the rate of nonlytic exocytosis was calculated based on the postsorted purity and postcultured live neutrophils not associated with C. neoformans.
Immunofluorescence staining of brain tissues
Mice were euthanized and perfused 3 h after i.v. infection by tail vein with 20 × 106 H99 cells, and the brain was removed for immunofluorescence staining. To avoid contamination of tissues by circulating fungi, perfusion was performed by injecting sterile saline (50 ml) into the left ventricle, and the right atrium was cut open to allow drainage during the procedure . The brain tissues of infected mice were prepared for frozen sections, as described previously . In brief, the tissues were removed and frozen in OCT compound. Frozen tissue blocks were cut on a cryostat microtome, and 5-μm sections were placed on coated glass slides. Tissue sections were fixed in 2% neutral buffered paraformaldehyde (PFA) for 10 min. Sections were then incubated with 2% goat serum in PBS, followed by incubation with rabbit-anti-mouse collagen IV (PA1–26148, Invitrogen), mouse-anti-cryptococcal polysaccharide (E1, a gift from Dr. F. Dromer, Paris, France), and rat-anti-mouse Ly6G (clone 1A8, Biolegend) at 4 °C overnight. After 3 washes, sections were incubated for 30 min with Alex Fluor 647 goat-anti-rabbit IgG (H + L) (A-21244, Invitrogen) to delineate brain microvasculature, Alexa Fluor 488 goat-anti-mouse IgG (H + L) (A-11001, Invitrogen) to identify C. neoformans, and Alexa Flour 555 goat-anti-rat IgG (H + L) (A-21434, Invitrogen) to identify neutrophils in the brain. The sections were rinsed and mounted with glycerol and checked under a confocal microscope.
Data are expressed as the mean ± SEM. An ANOVA was performed to establish equal variance, and a 2-tailed Student’s t test with Bonferroni correction was applied to determine statistical significance, which was defined as p < 0.05.
Nonlytic exocytosis of C. neoformans from neutrophils in vitro
Fungal capsule and viability influence nonlytic exocytosis
Roles of phagosome and actin in nonlytic exocytosis
As observed in Fig. 1, neutrophils expelled C. neoformans cells in 2 min. We therefore hypothesized that neutrophil actin polymerization may contribute to the nonlytic exocytosis of C. neoformans. In the expulsion of C. neoformans from macrophages, the actin flash was inversely correlated with nonlytic exocytosis . Therefore, we tested whether chemicals interfering with actin flashes play roles in nonlytic neutrophil exocytosis. As shown in Fig. 3, treatment with the actin polymerization inhibitor cytochalasin D significantly enhanced the nonlytic exocytosis of C. neoformans from neutrophils. Moreover, treatment with the Arp2/3 complex-mediated actin polymerization inhibitor wiskostatin also increased C. neoformans expulsion from neutrophils, although to a lesser extent. In contrast, treatment with jasplakinolide, a potent actin polymerization inducer, had a reduced effect on the nonlytic exocytosis. Collectively, these results show that classical WASP-Arp2/3 complex-mediated actin filament nucleation may play a role in the nonlytic exocytosis of C. neoformans from neutrophils.
Nonlytic exocytosis of C. neoformans from neutrophils in the brain vasculature
As the most commonly encountered clinical manifestation of cryptococcosis, cryptococcal meningoencephalitis is a devastating infectious disease. In AIDS patients, the mortality rate for cryptococcal meningoencephalitis was approximately 20% . Once arrested in the brain vasculature, C. neoformans may transmigrate across the BBB and proliferate in the brain parenchyma. Therefore, it is imperative to explore how immune cells respond to C. neoformans in the brain . Depletion of phagocytes, including macrophages and neutrophils, may impair the infiltration of C. neoformans into the brain . It has been established that macrophages [9, 10, 11] and monocytes  can expel intracellular C. neoformans. In the present study, we documented that nonlytic exocytosis of C. neoformans from neutrophils occurs in vitro and in vivo.
Neutrophils and macrophages originate from common myeloid progenitors. Neutrophils, however, are different from macrophages. First, neutrophils are considered key responders to infection. Once released into the bloodstream, C. neoformans transmigrate across the BBB within 6 h . Therefore, an immediate immune response is indispensable for the clearance or dissemination of C. neoformans brain infections. Indeed, intravascular clearance of C. neoformans is largely dependent on neutrophils . Second, the half-life of neutrophils is much shorter than that of macrophages. In ex vivo culture, neutrophils may die within 24 h. The average lifespan for circulating neutrophils, however, is as long as 5.4 days in vivo , raising the possibility that neutrophils may not be as fragile as expected. In the present study, neutrophils cocultured with endothelial cells and C. neoformans were observed and analyzed within several hours. As microbes may regulate the aging of neutrophils , we hypothesize that C. neoformans may affect the lifespan of neutrophils. Third, the neutrophil phagosome may be acidification independent . In the nonlytic exocytosis of C. neoformans from macrophages , phagosomal pH modulation was found to be associated with exocytosis. In neutrophils, phagosomal pH modification, either with vacuolar ATPase inhibitor bafilomycin A1 or ammonium chloride treatment, had little effect on the nonlytic exocytosis of C. neoformans. The effect of chloroquine on the occurrence of nonlytic exocytosis was largely related to the regulation of fungal viability rather than phagosomal acidification. Similar observations were observed for the capsule-deficient strains or heat-killed fungi.
Previously, we demonstrated that neutrophils may phagocytose arrested C. neoformans attached to the brain vasculature, which may help alleviate brain infections . In this study, we provide evidence that neutrophils expel intracellular C. neoformans in vitro and in the brain vasculature. We hypothesize that the role of neutrophils may be a double-edged sword, i.e., both protective and deleterious, during the course of cryptococcosis. On the one hand, neutrophils are important for killing  or removing C. neoformans arrested in the brain microvasculature; on the other hand, neutrophils carry C. neoformans and exocytose them in the brain vasculature, contributing to the brain infection.
Our study has some notable limitations. The nonlytic exocytosis of C. neoformans from monocytes and macrophages may originate from environmental interactions [38, 39]. In contrast, the evolutionary role of nonlytic exocytosis of C. neoformans from neutrophil is unknown. Moreover, neutrophils are heterogeneous with many different functions . Neutrophils with nonlytic exocytosis capabilities and the mechanisms warrant improved definitions.
Our results suggest that neutrophils adhering to brain vasculature extrude C. neoformans via nonlytic exocytosis, which is regulated by fungal virulence factors (viability and capsule) and neutrophils (phagosome and actin). The mechanisms behind the nonlytic exocytosis of C. neoformans from neutrophils in the brain vasculature require further study.
MZ designed and supervised the experiments; XY, XC and HF carried out the experiments; XY and HW carried out the statistical analyses, and edited figures and the manuscript. All the authors reviewed the paper. All authors read and approved the final manuscript.
This work was supported by a National Natural Science Foundation of China grant 81671563 (to MZ).
Ethics approval and consent to participate
All animal protocols were reviewed and approved by the Animal Care and Use Committee (IACUC) of Nanjing Medical University (1708004). The Experimental Animal Care and Use Guidelines from the Ministry of Science and Technology of China (MOST-2011-02) were strictly followed.
Consent for publication
The authors declare that they have no competing interests.
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